Notch signaling and bone fracture healing

描述（由申请人提供）：颅面和长骨缺损通常表现为延迟愈合或不愈合。虽然自体骨移植和骨形态发生蛋白被广泛用于治疗这种严重的损伤，但这些治疗方法有局限性。因此，临床仍然需要开发新的方法来增强骨再生。Notch信号通路调节成骨细胞增殖和成骨细胞分化。此外，Notch信号是许多组织伤口愈合的重要组成部分，损伤后Notch上调已被证明可以增强这些组织的再生。因此，本研究的长期目标是表明Notch信号也调节颅面和长骨组织的再生，通过临床应用的组织工程策略上调Notch信号可以促进颅面骨再生。以下三个具体目标补充了NIDCR 2009-2013年战略计划（草案）目标1-5，即“通过生物、生物工程和生物材料研究方法促进患病或受损口腔和颅面组织和器官的重建和再生。”对于第一个特定的目的，我们将表征Notch信号在颅骨缺损和胫骨骨折愈合。缺口报告小鼠将经历3毫米直径的颅骨缺损或双侧胫骨骨折。将小鼠在损伤前和损伤后1、2、5、10、15、20和40天处死，进行qPCR和免疫组织化学分析。在具体目标2中，我们将确定Notch信号对于成功的颅骨和胫骨骨再生的关键重要性。具有cre调控的典型缺口抑制剂dnMAML表达的转基因小鼠将经历3mm的颅骨缺损或双侧胫骨骨折，并且将局部给予腺- cre以永久抑制notch信号（腺- lacz控制）。小鼠将于损伤后5、10、20和40天处死，进行|JCT、组织学和生物力学测试。为了最终的具体目标，我们将开发一种缺口组织工程疗法来增强颅骨骨再生。作为概念验证，我们将在野生型小鼠的3mm颅骨缺陷中施用腺- nicd（腺- lacz对照），以瞬时上调Notch。然后，我们将Notch配体Jagged-1吸附到骨传导生物材料上，并将这些组织工程构建体植入3mm的颅骨缺损。本研究的目的是：1)探讨Notch信号通路在颅面骨愈合中的作用；2)表明Notch操作在临床上可以促进颅面骨再生。

DESCRIPTION (provided by applicant): Craniofacial and long bone defects often exhibit delayed or non-union healing. While autogenous bone grafts and bone morphogenetic proteins are widely used to treat such severe injuries, these therapies have limitations. Therefore, a clinical need persists for the development of new methods to enhance bone regeneration. The Notch signaling pathway regulates osteoprogentor proliferation and osteoblast differentiation. Additionally, Notch signaling is an essential component for wound healing in many tissues, and Notch upregulation following injury has been shown to enhance regeneration of those tissues. Therefore, the long-term objectives of this study are to show that Notch signaling also regulates regeneration of craniofacial and long bone tissue, and that upregulation of Notch signaling through a clinically-applicable tissue engineering strategy can enhance craniofacial bone regeneration. The following three specific aims complement the NIDCR's strategic plan for 2009-2013 (draft) objective 1-5, which is to 'Facilitate reconstruction and regeneration of diseased or damaged oral and craniofacial tissues and organs through biological, bioengineering and biomaterials research approaches.' For the first specific aim we will characterize Notch signaling during calvarial defect and tibial bone fracture healing. Notch reporter mice will undergo a 3 mm diameter calvarial defect or bilateral tibial fractures. Mice will be sacrificed pre-injury and at 1, 2, 5, 10, 15, 20 and 40 days post-injury for qPCR and immunohistochemistry analysis. In specific aim 2, we will determine the critical importance of Notch signaling for successful calvarial and tibial bone regeneration. Transgenic mice with Cre-regulated expression of the canonical notch inhibitor dnMAML will undergo a 3 mm calvarial defect or bilateral tibial fractures and adeno-Cre will be locally administered tp permanently inhibit Notch signaling (adeno-LacZ control). Mice will be sacrificed at 5, 10, 20 and 40 days post-injury for |JCT, histology, and biomechanical testing. For the final specific aim we will develop a Notch tissue engineering therapy to enhance calvarial bone regeneration. As a proof of concept, we will administer adeno-NICD (adeno-LacZ control) to 3 mm calvarial defects in wild type mice to transiently upregulate Notch. We will then adsorb Notch ligand, Jagged-1 onto an osteoconductive biomaterial and implant these tissue engineered constructs in 3 mm calvarial defects. The purpose of this study is to 1) investigate the role(s) of the Notch signaling pathway in craniofacial bone healing, and 2) show that Notch manipulation can clinically enhance craniofacial bone regeneration. Public Health Relevance: Craniofacial and long bone defects often exhibit delayed or non-union healing. While autogenous bone grafts and bone morphogenetic proteins are widely used to treat such severe injuries, these therapies have limitations. Therefore, a clinical need persists for the development of new methods to enhance bone regeneration. The purpose of this study is to 1) investigate the role(s) of the Notch signaling pathway in craniofacial bone healing, and 2) show that Notch manipulation can clinically enhance craniofacial bone regeneration.